Published on Web Date: March 16, 2010

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Discovery of NVP-LDE225, a Potent and SelectiveSmoothened AntagonistShifeng Pan,*,† Xu Wu,† Jiqing Jiang,† Wenqi Gao,† Yongqin Wan,† Dai Cheng,† Dong Han,†Jun Liu,† Nathan P. Englund,† Yan Wang,† Stefan Peukert,‡ Karen Miller-Moslin,‡ Jing Yuan,‡Ribo Guo,‡ Melissa Matsumoto,‡ Anthony Vattay,‡ Yun Jiang,‡ Jeffrey Tsao,‡ Fangxian Sun,†AnneMarie C. Pferdekamper,† Stephanie Dodd,‡ Tove Tuntland,† Wieslawa Maniara,‡Joseph F. Kelleher, III,‡ Yung-mae Yao,‡ Markus Warmuth,‡ Juliet Williams,‡,§ andMarion Dorsch‡

†Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, California 92121, and‡Novartis Institute for Biomedical Research, 250 Massachusetts Avenue, Cambridge, Massachusetts 02139

ABSTRACT The blockade of aberrant hedgehog (Hh) signaling has shownpromise for therapeutic intervention in cancer. A cell-based phenotypic high-throughput screen was performed, and the lead structure (1) was identified as aninhibitor of the Hh pathway via antagonism of the Smoothened receptor (Smo).Structure-activity relationship studies led to the discovery of a potent and specificSmoothened antagonist N-(6-((2S,6R)-2,6-dimethylmorpholino)pyridin-3-yl)-2-methyl-40-(trifluoromethoxy)biphenyl-3-carboxamide (5m, NVP-LDE225), whichis currently in clinical development.

KEYWORDS Hedgehog signaling pathway, Smoothened, medulloblastoma

Smoothened (Smo) is a 7-pass transmembrane proteinthat functions as the key activator of the hedgehog(Hh) signaling pathway.1 While Hh signaling is tightly

controlled during cellular proliferation, differentiation, andembryonic morphogenesis,2,3 aberrant pathway activationhas been linked to tumorigenesis in several cancers.4-6 Thispathway can be stimulated by one of three Hh ligands: sonichedgehog (Shh), Indian hedgehog (Ihh), or desert hedgehog(Dhh). Upon binding of Hh ligand to the 12-pass transmem-brane protein Patched (Ptch), the repression of Smo by Ptchis relieved. Activated Smo then initiates a downstreamsignaling cascade leading to the activation of transcriptionfactors of the Gli family. Strong genetic evidence (mutationsin Ptch, Smo, SUFU, or Gli) links up-regulated pathwayactivity to tumor formation in cancers such as basal cellcarcinoma and medulloblastoma.7-9 Furthermore, Hhsignaling has been shown to play a role in tumorigenesisof various other cancers such as pancreatic, prostate,lung, colorectal, bladder, and ovarian. Thus, inhibitionof Hh signaling is an attractive approach for anticancertherapy.10,11

Cyclopamine (Figure 1), a naturally occurring alkaloid,was the first Smo antagonist to be reported in the litera-ture.12 It has been shown to inhibit Hh signaling and toinduce the remission of medulloblastoma in a transgenicmouse model.9 Several other synthetic small molecule Smoantagonists have been described in recent years.10,11 TheSmo inhibitor GDC-0449 (Figure 1), recently reported byGenentech, has shown promising phase I clinical results inadvanced basal cell carcinoma patients.13 A cyclopamine

derivative IPI-926 (Figure 1) has also entered clinical deve-lopment recently. Herein, we report our efforts in this area,which led to the discovery of N-(6-((2S,6R)-2,6-dimethylmor-pholino)pyridin-3-yl)-2-methyl-40-(trifluoromethoxy)biphenyl-3-carboxamide (5m, NVP-LDE225), a potent and selectiveSmo antagonist currently in phase I clinical trials.

A high-throughput cell-based screen of in-house diversitycombinatorial libraries (10K compounds generated by solid-phase synthesis), using a reporter gene assay in amouse cellline (TM3) stably transfected with a Hh-responsive Gli luci-ferase construct and stimulated by Shh protein, led to theidentification of a class of biphenyl carboxamides, such ascompound 1 (Figure 2), as Hh signaling inhibitors. To con-firm Smo as the target for these screening hits, a Gli IC50 shift

Figure 1. Structures of cyclopamine, GDC-0449, and IPI-926.

Received Date: February 10, 2010Accepted Date: March 10, 2010

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assay was employed.14 A known Smo agonist, Ag1.5,15 wasused at two different concentrations (1 and 25 nM) to induceHh pathway activation in the reporter gene assay. A shift toa higher IC50 at the higher concentration of Ag1.5 wasexpected for a competitive inhibitor. Compound 1 resultedin IC50 values of 0.17 and 1.13 μM, respectively, under theseconditions, corresponding to a 6.5-fold IC50 shift, suggestingits direct interaction with the Smo receptor. Fluorescencebinding assays, with both human and mouse Smo, viacompetition of BODIPY-cyclopamine were also employedin our lead optimization effort to ensure cross-reactivity. Asystematic study of structure-activity relationships (SARs)was carried out in three regions (A-C) of compound 1(Figure 2).

The general synthetic route used to prepare these com-pounds is illustrated in Scheme 1. It primarily involved twotransformations, amide bond formation and Suzuki cross-coupling, from commercially or readily available startingmaterials. This versatile combination of reaction sequencesallowed for the rapid investigation of SARs in all threeregions. The key SAR results for select analogues within thisseries are summarized in Table 1.

Our initial focus was on exploring modifications to regionA, which in compound 1 features an electron-rich 1,4-diaminophenyl moiety. Replacement of this moiety wasdesirable as it could potentially form quinone-like reactiveintermediates upon metabolic activation, thereby leading topromiscuous toxicities. Tomitigate this risk, several differentapproaches were employed aiming to reduce the electrondensity of this moiety. The introduction of electron-with-drawing groups at the R2 position, such as-F (5b) and-CF3(5c), led tomarginal increases in activity. The replacement ofphenyl with a more electron-deficient pyridine (compound5d) was also tolerated. However, an attempt to move themorpholine nitrogen one carbon atom away from its directattachment to the phenyl ring was unsuccessful (compound5e). At the R3 position, a piperidinyl substituent producedcomparable activity to morpholino (compounds 5f vs 5d).Interestingly, the position of the nitrogen in the pyridinemoiety was critical for the inhibitory activity. The regioiso-meric pyridinyl analogue (compound 5g) led to a significantloss of activity. An appreciable enhancement of activity wasachieved upon introduction of cis-dimethyl groups onto themorpholine ring (compound 5h).

Substituents on the central phenyl ring (region B) playedan important role in modulating Hh signaling inhibitoryactivity. Early SARs suggested that a small hydrophobicgroup, such as -Me (compound 5a), was favored at the4-position. A breakthrough in increasing potency was rea-lized upon migrating the methyl group from the 4- to the2-position (compound 5i). Similarly, a 2-chloro substituent

gave comparable activity (compound 5j). A detailed surveyof substitutions in region C led to the identification of a fewpreferred substituents at the R4-position, including -CN(5a-j), -OMe (5k), -CF3 (5l), and -OCF3 (5m). As ourprimary assays to evaluate these compounds used a mousecell line (TM3), select compounds were tested in fluores-cence binding assays with both mouse and human Smo toensure desired cross-reactivity between species, throughcompetition of BODIPY-cyclopamine, a fluorescent-labeledcyclopamine derivative. We were encouraged by the obser-vation that the IC50 values for mouse and human Smo werehighly consistent for the tested compounds.

In a traditional lead optimization approach, pharmaceu-tical properties, in vitro ADMET and in vivo pharmacoki-netics (PKs), were evaluated for compounds meeting thepotency and selectivity criteria. On the basis of overallfavorable properties, compound 5m (NVP-LDE225) waschosen for further in vitro and in vivo testing.

Compound 5m is highly bound to mouse, rat, and humanplasma proteins (>99%) and moderately bound to dog andmonkey plasma proteins (77 and 85%, respectively). Thecompound has high permeability in the PAMPA assay(calculated dose fraction absorbed estimated to be 90.8%in man). Compound 5m shows good oral bioavailabilityranging from 69 to 102% in preclinical species when dosedin solution (Table 2). The systemic plasma clearance (CL) islow (mouse, rat, and monkey) to moderate (dog) relative torespective hepatic blood flow, while the volume of distribu-tion (Vss) is moderate (mouse and rat) to high (dog andmonkey) as compared to total body water. The meanresidence time (MRT) ranges from 3.04 to 6.81 h. The abilityto penetrate the blood-brain barrier is considered favorablewhere the brain/plasma AUC0-inf ratio is moderate (0.57).Therefore, good exposures are predicted in medulloblastoma,

Figure 2. Structure of 1, a screening hit.

Scheme 1. General Synthetic Schemea

aReagents and conditions: (a) Anilines or aminopyridines, HATU, Et3N,DMF. (b) Boronic acids, cat. Pd(PPh3)4, Na2CO3, DME-H2O.

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a type of brain tumor, which is a potential clinical indicationfor Smo antagonists.

Compound 5m is aweakbasewith ameasured pKa of 4.20and exhibits relatively poor aqueous solubility. In contrast tohigh oral bioavailability in rats when administered as asolution in PEG300/dextrose water, only marginal drug ex-posure (oral bioavailability 3%)wasobtainedwhen a crystal-line free base was dosed as a suspension in 0.5% sodiumcarboxymethyl cellulose. To increase the drug oral exposure,a stable crystalline diphosphate salt with an improved dis-solution rate was developed. Following oral administrationin the same suspension formulation, the absolute oralbioavailability was increased to 48%.

Ptchþ/-p53-/- mice, which have been shown to sponta-neously develop medulloblastomas,9,16,17 have been effec-tively used to evaluate in vivo antitumor efficacy of Smoantagonists, both directly in the transgenic model16 and inallograft models derived from the Ptchþ/-p53-/- medullo-blastoma tumors.9,14,18,19 Given that 5m displayed both a

favorable PK profile across preclinical species and brainexposure upon oral administration, we elected to assess itsantitumor efficacy using both subcutaneous and orthotopicPtchþ/-p53-/- medulloblastoma allograft models.

In the subcutaneous Ptchþ/-p53-/- medulloblastoma allo-graft mouse model, 5m demonstrated dose-related antitumoractivity after 10 days of oral administration of a suspen-sion of the diphosphate salt (Figure 3). At a dose of 5 mg/kg/day qd, 5m significantly inhibited tumor growth, correspond-ing to a T/C value of 33% (p < 0.05 as compared to vehiclecontrols).Whendosedat10and20mg/kg/dayqd,5mafforded51 and 83% regression, respectively. Mouse Gli1 mRNA ex-pression levels were utilized as a pharmacodynamicmarker tolink the antitumor effects of 5m to inhibition of Hh pathwayactivity in this model. Tumors were harvested at different timepoints following a single dose of 5m at 5, 10, or 20mg/kg, andlevels of Gli1 mRNA expression were analyzed by real-timepolymerase chain reaction (PCR) (Figure 4). As illustrated inFigure 4, a dose- and time-dependent inhibition of Gli1 mRNAlevels was observed in the Ptchþ/-p53-/- medulloblastomaallograft model. Gli1 mRNA inhibition correlated with tumorand plasma exposure of 5m. Interestingly, a time delay wasobserved between achieving peak concentrations of drug andmaximum inhibition of Gli1 levels, presumably due to the timerequired to inhibit downstream Gli1 transcription and the Gli1mRNA turnover time.

An orthotopic Ptchþ/-p53-/- medulloblastoma allograftmodel was also established by implanting tumor cellsharvested from Ptchþ/-p53-/- mice stereotaxically into thefrontal cortex of nude mice. Treatment was initiated on day

Table 1. IC50 Values for Select Compounds in the TM3-Gli-Luc IC50 Shift Assay and Mouse (m) and Human (h) Smo Fluorescence BindingAssays

IC50 (nM)

R1 R2 R3 R4 X Y 1 nM Ag1.5a,b 25 nM Ag1.5a,b mBdga,c hBdga,c

5a 4-Me H A CN CH CH 43 409

5b 4-Me F A CN CH CH 17 256

5c 4-Me CF3 A CN CH CH 22 246

5d 4-Me H A CN CH N 14 271 80 124

5e 4-Me H B CN CH CH 247 4385

5f 4-Me H C CN CH N 14 334

5g 4-Me H C CN N CH 751 3914

5h 4-Me H D CN CH N 5 71 5 12

5i 2-Me H D CN CH N 0.6 7.3 0.5 2

5j 2-Cl H D CN CH N 0.7 14

5k 2-Me H D OMe CH N 0.6 10

5l 2-Me H D CF3 CH N 0.8 7.9 3.1 11

5m 2-Me H D OCF3 CH N 0.6 8 1.3 2.5a See the Supporting Information for detailed assay descriptions. bReporter gene assay in a TM3 luciferized cell line with activation of the pathway

using varying concentrations of the Hh agonist Ag1.5. c Smo binding assay utilizing the displacement of BODIPY-cyclopamine.

Table 2. In Vivo Pharmacokinetics among Speciesa

CL (mL/min/kg) Vss (L/kg) MRT (h) F (%)

mouse 10.4 1.9 3.04 69

rat 6.0 1.5 4.17 83

dog 17.1 6.99 6.81 101

monkey 14.8 3.4 3.83 102a The compound was administered as a solution in PEG300/5%

dextrose in water (75:25 v/v) for mouse and rat or in 20% Captisol fordog and monkey.

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17 following tumor implantation in mice demonstratingestablished tumors as determined by MRI imaging. Treatedanimals were dosed at 40 mg/kg/day po bid, and tumor sizewas assessed by MRI after 4 days of dosing (Figure 5). Asillustrated in Figure 5, the established tumors grew signifi-cantly in the vehicle-treated animals over the 4 day treat-ment period (þ1230 ( 210% as compared to baseline). Incontrast, 5m treatment clearly slowed tumor growth relativeto vehicle-treated animals (þ124 ( 37% as compared tobaseline). A separate study performed with a contrast agentdemonstrated that the blood-brain barrier remained intactin this model following implantation of tumor cells.20

Together, these studies suggest that 5m successfully pene-trates the blood-brain barrier in tumor-bearing animalsand results in tumor growth inhibition after 4 days oftreatment.

As part of the pharmaceutical developability assessment,compound 5mwas run through a series of preclinical safetyassays. The IC50 values for 5m for the major human CYP450drug metabolizing enzymes was greater than 10 μM. Inaddition, 5m did not exhibit time-dependent CYP inhibitionnor induction of CYP3A4, suggesting low potential fordrug-drug interactions. In an automated hERG patch clamp

assay, it showed an IC50 value of greater than 30 μM. Com-pound 5m was negative in tests for genotoxicity (Ames andmicronucleus tests). The selectivity of compound 5m wasevaluated by screening against a large panel of receptors, ionchannels, transporters, kinases, and proteases. No appreci-able activities (i.e., IC50<10 μM)were identified, suggestingits low potential for off-target effects.

In summary, a novel chemical series identified via high-throughput screening, the biphenyl-3-carboxamides, was op-timized for Smo antagonism, selectivity, safety, and PKs todiscover the clinical candidate 5m. Treatment with 5m in asubcutaneous Ptchþ/-p53-/-medulloblastoma allograft mousemodel led todose-related tumorgrowth inhibition,with tumorregression observed in the higher dosing groups. The abilityof 5m to penetrate the blood-brain barrier and to inhibittumor growth in brain was demonstrated in an orthotopicPtchþ/-p53-/-medulloblastoma allograftmousemodel. Thiscompound is now in phase I clinical trials, and its clinical PK,efficacy, and safety are currently under evaluation.

Figure 3. Antitumor activity upon treatment with 5m diphos-phate salt or vehicle in a Ptchþ/-p53-/- medulloblastoma subcu-taneous allograft model in nude mice. Treatment started on day 8postimplantation (5 million cells/animal). Compound 5m wasadministered po at 5 (filled triangle), 10 (open triangle), and 20(filled diamond) mg/kg/day qd for 13 days total. All doses areexpressed as free base equivalents. Vehicle control (open square)of 5m: 0.5% methylcellulose and 0.5% Tween 80 in water. Alltreatment groups consisted of eight animals. The vehicle groupwas taken down 7-9 days after treatment due to excessive tumorsize (greater than 10% of mouse body weight). The body weightchange observed was <(5% for all treated groups.

Figure 4. Gli1mRNA inhibition (open circle), tumor PK (filled squares), and plasma PK (filled triangles) in Ptchþ/-p53-/-medulloblastomamodel after treatment with 5m. Plasma and tumors were harvested at 4, 8, 16, and 24 h after a single oral dose of 5m at 5, 10, or 20 mg/kg.Gli1 mRNA levels were analyzed by real-time PCR and normalized to β-actin expression. Data shown are percent inhibition relative tovehicle-treated control tumors. Compound 5m concentrations were determined in plasma and tumor by LC-MS/MS. N= 3 per time point.

Figure 5. Antitumor activity in an orthotopic Ptchþ/-p53-/- me-dulloblastoma allograft model in nude mice upon treatment with5m diphosphate salt dosed at 40 mg/kg/day po bid or vehicle atequal dose volume. Athymic nude mice were implanted with100000 tumor cells 17 days before the start of dosing. Dailytreatment (vehicle or 5m) was initiated on day 0. Eight animalswere enrolled into each group. MRI scans were performed atbaseline and day 4 postinitiation of treatment. The bar graphrepresents the group mean ( SEM, with crosses indicating datafrom individual animals. At each time point, a representative 3Drendering and paired MRI are shown of an animal representativeof the group mean. To enable longitudinal comparisons, theselected MRI is from the same animal over time point.

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SUPPORTING INFORMATION AVAILABLE Procedures forthe preparation of 5m, analytical data, and procedures and meth-ods for in vitro and in vivo assays. This material is available free ofcharge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATIONCorresponding Author: *To whom correspondence should beaddressed. Tel: 858-812-1621. E-mail: span@gnf.org.

Present Addresses: § Cancer Research Technology, GowerStreet, London WC1E 6BT, United Kingdom

ACKNOWLEDGMENT We thank Lucas Westling for analyticalsupport and Dr. Rosalind Segal and Dr. Andrew Kung (Dana-FarberCancer Institute, Boston, MA) for providing Ptchþ/-p53-/- tumors.

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(20) See the Supporting Information for full details.